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DNA triplex formation by base, backbone, and sugar-ring modified oligonucleotides with HIV targets.

机译：通过具有HIV靶标的碱基，主链和糖环修饰的寡核苷酸形成DNA三链体。

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摘要

Triplex forming oligonucleotides (TFOs) bind to polypurine tracts in the major groove of DNA. This thesis describes studies on TFOs that target the env- and gag-genes of HIV-1 proviral DNA. Interactions between nuclease resistant, 5′-psoralen conjugated, chimeric methylphosphonate oligodeoxyribo- or oligo-2′ -O-methylribo-triplex forming oligomers (psTFOs), and the purine tract of env-DNA were investigated by gel mobility shift assays. A pyrimidine, chimeric methylphosphonate psTFO composed of thymidine and 5-methyl-2 ′-deoxycytidine, formed a stable triplex with env-DNA (apparent Kd = 1.3 μM). The apparent Kd of another chimeric psTFO decreased to 400 nM when four thymidines were replaced with 5-propynyl-2′ -deoxyuridines. The apparent Kd of the all-phosphodiester counterpart TFOs decreased to 50 nM. A chimeric oligo-2′-O-methylribopyrimidine formed a stable triplex (apparent Kd = 1.4 μM). Antiparallel A/G oligomers and parallel or antiparallel T/G oligomers did not form triplexes.; Thermal denaturation experiments were used to investigate interactions of psTFOs with env-DNA or gag-DNA targets. Triplex stability of a deoxy-env-psTFO containing 5-methylcytosines and thymines decreased with increasing pH. Replacement of 5-methylcytosines with 8-oxo-adenines reduced pH dependence but lowered triplex stability. A 2′-O-methyl-env-psTFO containing uracil and cytosine did not form a triplex at pH 7.5. Replacement of cytosines with 5-methylcytosines increased triplex stability, and greater stability was achieved by selective replacement of uracils with thymines. Substitution of contiguous 5-methylcytosines in a deoxy-gag-psTFO with 8-oxo-adenines reduced pH dependence and increased triplex stability. Triplexes formed by 2′-O-methyl-gag-psTFOs did not show enhanced stability. Replacement of the TFO 3′-terminal phosphodiester with a methylphosphonate increased nuclease resistance of deoxy- and 2′-O-methyl-TFOs.; In vitro transcription experiments were performed to determine the effect of psTFOs on transcription of pGEM®-3Z plasmids containing the env-DNA insert. Two env-specific and two gag-specific psTFOs were investigated. Non-specific inhibition of transcription was observed in the presence all psTFOs, with and without UV irradiation. Upon irradiation, specific inhibition of synthesis of the full length transcript was observed in the presence of env-specific psTFOs. Thus, all four psTFOs demonstrated non-specific inhibition of transcription of the env-DNA target. Specific inhibition of transcription was only observed when the env-specific psTFOs were covalently crosslinked to env-DNA.
机译：形成三链体的寡核苷酸（TFO）与DNA主沟中的聚嘌呤束结合。本文描述了针对以HIV-1前病毒DNA的 env -和 gag 基因为目标的TFO的研究。耐核酸酶的5 '-补骨脂素缀合的，嵌合的甲基膦酸酯寡脱氧核糖-或oligo-2 '-O-甲基核糖三链形成寡聚物（psTFOs）与嘌呤束之间的相互作用通过凝胶迁移率变动分析研究了 env -DNA的表达。嘧啶，由胸苷和5-甲基-2 '-脱氧胞苷组成的嵌合甲基膦酸酯psTFO与 env -DNA形成稳定的三链体（表观Kd = 1.3μM）。当四个胸苷被5-丙炔基-2 '-脱氧尿苷替代时，另一种嵌合psTFO的表观Kd降低至400nM。全磷酸二酯对应的TFO的表观Kd降至50 nM。嵌合的oligo-2 ' -O-甲基核糖嘧啶形成稳定的三链体（表观Kd = 1.4μM）。反平行A / G低聚物和平行或反平行T / G低聚物不形成三链体。通过热变性实验研究了psTFO与 env -DNA或 gag -DNA靶标的相互作用。含5-甲基胞嘧啶和胸腺嘧啶的脱氧- env -psTFO的三元组稳定性随pH的升高而降低。用8-氧代腺嘌呤替代5-甲基胞嘧啶可降低pH依赖性，但降低三链体稳定性。含尿嘧啶和胞嘧啶的2 ' -O-甲基- env -psTFO在pH 7.5时未形成三链体。用5-甲基胞嘧啶替代胞嘧啶可增加三链体的稳定性，并且通过用胸腺嘧啶选择性替代尿嘧啶可达到更高的稳定性。用8-氧代-腺嘌呤取代连续的5-甲基胞嘧啶取代脱氧- gag -psTFO可降低pH依赖性并增加三链体稳定性。由2 ' -O-甲基- gag -psTFO形成的三链体未显示出增强的稳定性。用甲基膦酸酯代替TFO 3 '-末端磷酸二酯增加了脱氧-和2 ' -O-甲基-TFO的核酸酶抗性。进行体外转录实验以确定psTFOs对含有 env -DNA插入片段的pGEM ® -3Z质粒转录的影响。研究了两个 env 特异性和两个 gag 特异性psTFO。在有和没有紫外线照射下，在所有psTFOs的存在下均观察到转录的非特异性抑制。照射后，在 env 特异性psTFOs存在下，观察到全长转录物的合成被特异性抑制。因此，所有四个psTFOs都显示了 env -DNA靶标的转录的非特异性抑制。仅当 env 特异性psTFOs与 env -DNA共价交联时才观察到转录的特异性抑制。

著录项

  • 作者

    Cassidy, Rachel Ann.;

  • 作者单位

    The Johns Hopkins University.;

  • 授予单位 The Johns Hopkins University.;
  • 学科 Biophysics General.; Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2004
  • 页码 134 p.
  • 总页数 134
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物物理学;生物化学;
  • 关键词

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